Dendritic BC1 RNA Inhibits Translation by Targeting Initiation Factors eIF4A and PABP.

Abstract

The cellular and molecular basis of neuronal plasticity is closely associated with the regulation of local protein synthesis in dendrites. Postsynaptic local protein synthesis requires mRNA to be transported from the cell body of the neuron to its dendrites. Following physiological stimulation of an individual synapse, mRNAs at that synapse are translated in response to that external stimulus, thus contributing to input specific synaptic plasticity. Rodent BC1 RNA is a small non-translatable RNA in that is highly abundant in dendrites. Previously, BC1 RNA has been shown to inhibit translation and to interact with eukaryotic initiation factors 4A (eIF4A) and poly (A) binding protein (PABP). In this thesis, I examine the underlying functional mechanisms between BC1 RNA and its two binding factors. Biochemically, BC1 RNA binds to eIF4A with a calculated Kd of 0.2 nM. BC1 RNA competes effectively against RNA duplex substrate for eIF4A binding with an IC50 of 8.5 nM. eIF4A is an RNA helicase that hydrolyzes ATP to unwind mRNA secondary structure during translation initiation. Functionally, BC1 RNA inhibits eIF4A-dependent RNA duplex unwinding and the helicase stimulator eIF4B is not able to overcome this inhibition. In ATPase assays, BC1 RNA stimulates eIF4A-dependent ATP hydrolysis. Primate BC200 RNA inhibits eIF4A-depedent RNA duplex unwinding and stimulates its ATPase activity in a similar manner as BC1 RNA. Data indicate that eIF4A-dependent helicase activity is uncoupled from its ATPase activity by both BC RNAs. UV cross-linking assays demonstrate that eIF4A and eIF4B synergistically enhance each other to bind BC1 RNA. These assays also show ATP and ADP increase BC1-eIF4A cross-linking efficiency by 10 and 4-fold, respectively. Data suggest that eIF4B and adenosine nucleotides are involved in the modulation of the BC1-eIF4A interaction. PABP is a critical initiation factor that binds to the poly (A) tail of the mRNA. Results of electrophoresis mobility shift assays (EMSA) indicate that BC1 RNA and the repression competent 3’ BC1 domain interact specifically with the RNA Recognition Motif (RRM) 1+2 of PABP. PABP binds to BC1 RNA with an estimated Kd of 8 nM, but does not reduce BC1 RNA inhibition of eIF4A-dependent RNA duplex unwinding. In HEK293 cells, BC1 RNA inhibits translation of 5’ secondary structure containing chloramphenicol acetyltransferase (CAT) mRNA with or without a poly (A) tail. Data demonstrate a direct link between BC1 RNA repressed translation and its two binding factors in vivo. My data suggest that the mechanism by which dendritic BC1 RNA inhibits translation is to target the catalytic activity of eIF4A and to interact specifically with PABP.